APOB is a potential prognostic biomarker in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is significantly associated with adverse prognostic outcomes. The development and progression of different types of human tumors are significantly influenced by APOB. Nevertheless, the significance and pathomechanisms of APOB in HCC have not been conclusively determined. We assessed APOB expression levels in HCC using three publicly available databases of TIMER2.0, UALCAN and Human Protein Atlas. To identify the biological function of APOB, we conducted enrichment analysis via LinkedOmics. Moreover, UALCAN was employed to assess the relationship between APOB expression and clinicopathological features among HCC patients. Additionally, the Kaplan–Meier plotter was utilized to investigate the prognostic relevance of APOB in HCC. To explore potential regulatory ncRNAs that could bind to APOB, we utilized StarBase and GEPIA. Furthermore, the correlation between APOB expression and immune cell infiltration, as well as immune checkpoint genes, was investigated using Spearman's correlation analysis in TISIDB, GEPIA, and TIMER2.0. The findings of our investigation showed a notable decrease in the expression levels of APOB among individuals diagnosed with HCC. Moreover, a noteworthy correlation was observed between the expression of APOB and immune checkpoint genes, alongside the occurrence of immune cell infiltration. The levels of APOB expression in HCC tissues also showed correlations with various clinicopathological features. According to Cox regression analysis, decreased APOB expression emerged as a potential autonomous predictor for OS, RFS, DSS, and PFS among HCC patients. Furthermore, we identified six potential pathways associated with non-coding RNA (ncRNA) as the most promising pathway for APOB in HCC. Our results illuminate the possible involvement of APOB in HCC and offer understanding into its governing mechanisms and medical importance. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-024-00877-6.


Introduction
Hepatocellular carcinoma (HCC) is a prevalent cancer recognized for its unfavorable outlook due to its high fatality rate and complex origins [1,2].It holds the second position globally in terms of deaths related to cancer [3,4].Despite the progress in treatment options for HCC, such as radiofrequency ablation, transplantation, transarterial chemoembolization, and surgical resection, the survival rate remains disappointingly low [5][6][7][8].Hence, there is an urgent need to create efficient measures for HCC that can enhance the quality of life and boost the chances of survival [9][10][11].
Among the apolipoprotein family members, APOB consists of tiny particles that carry dietary lipids through the bloodstream from the intestines to the liver [22,23].APOB primarily produces two isoforms of proteins, namely apoB-100 and apoB-48 [24,25], which are predominantly present in the serum.It is worth noting that previous research has shown a connection between APOB and diverse forms of cancer, such as gallbladder cancer [26,27], low-grade glioma [28], non-small cell lung cancer [29,30], and primary small cell carcinoma of the esophagus [31].Furthermore, research conducted by Lee and colleagues indicated that individuals with HCC who have a deactivated APOB gene experience worse results [32].According to another research, it was proposed that the metabolic reprogramming of HCC could occur due to a substantial decrease in APOB caused by hypermethylation [33].Nevertheless, the specific cause of this connection remains unknown.
Initially, we assessed the concentrations of APOB in HCC.Subsequently, we performed an analysis of clinical parameters associated with APOB and conducted survival analysis.Moreover, the investigation of APOB regulation in HCC involved the studing non-coding RNA (ncRNA), such as miRNAs and lincRNAs.Lastly, the investigation of the relationships between APOB levels and immune cell infiltration, as well as immune checkpoints, was also conducted in HCC.Our results suggest that down-regulation of APOB, facilitated by ncRNAs, correlates with adverse prognostic outcomes and the infiltration of immune cells among HCC patients.

TIMER2.0
The TIMER2.0 tool is a comprehensive software tool (http:// timer.comp-genom ics.org/) that facilitates the systematic analysis of immune infiltrates across various cancer types [34][35][36].It provides estimations of immune infiltrate abundances through the utilization of multiple immune deconvolution methods.This enables users to generate visually appealing and informative figures, allowing for a comprehensive exploration of genomic characteristics, clinical attributes, and tumor immunological aspects.In the context of HCC, TIMER2.0 was employed to evaluate the prevalence of tumor infiltrates and establish relationships between APOB levels and the expression of immune checkpoints, as well as the levels of immune cell infiltration.

LinkedOmics
LinkedOmics enables the execution of multi-omics analysis on TCGA datasets (http:// www.linke domics.org/ login.php) [37,38].The TCGA-LIHC project was selected for analysis, encompassing a cohort of 371 HCC patients (Data type: RNA-seq; Date: 01/28/2016).The genes that showed differential expression and were associated with APOB were acquired from the Link-Finder module.The Pearson correlation coefficient was utilized for evaluation, and the results were represented using heat maps and volcano plots.Additionally, gene set enrichment analysis (GSEA) was conducted using the LinkInterpreter module for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).

ULACAN
The UALCAN platform offers comprehensive analyses of transcriptional data derived from The Cancer Genome Atlas (TCGA) (http:// ualcan.path.uab.edu/ index.html) [39,40].This database was used to determine APOB levels correlated with various clinical and pathological parameters (sex, nodal metastasis status, and tumor grade cancer stage) of HCC.The APOB mRNA data is quantified as Transcript per million.Disparities in APOB expression between two groups were assessed through the utilization of Welch's T-test [41].

Human protein Atlas
The Protein Atlas for Humans offers immunohistochemical data on the expression of proteins in 20 different types of cancer, with each type consisting of 12 distinct tumors (https:// www.prote inatl as.org) [42,43].This can be utilized for the

APOB levels are decreased in HCC patients
Initially, the TIMER 2.0 was employed to investigate the level of APOB mRNA in human tumors.Figure 1A showed a decrease in APOB levels in cholangiocarcinoma (CHOL), liver HCC (LIHC), and breast invasive carcinoma (BRCA) compared to normal tissues.Moreover, analysis of the UALCAN databases revealed a substantial decrease in APOB mRNA expression in HCC patients relative to normal tissues (Fig. 1B).
Next, the Human Protein Atlas was utilized to assess the protein levels of APOB in HCC.In normal liver tissues, APOB protein was undetectable, as depicted in Fig. 1C, while HCC tissues exhibited detectable but minimal levels.

Enrichment analyses of APOB Co-expressed Genes in HCC
To explore the biological functions and pathways of APOB in HCC, there was a positive correlation between APOB and 3843 genes, whereas APOB exhibited a negative correlation with 8125 genes (Fig. 2A).The heat maps illustrating the top 50 genes associated with APOB are presented in Fig. 2B and Fig. 2C.The examination of biological processes (BP) revealed that APOB's co-expressed genes were significantly concentrated in the metabolic process of fatty acids, immediate inflammatory reaction, reaction to foreign substances, control of synapse structure or activity, and control of cytoskeleton organization (Fig. 2D).The analysis of cellular components (CC) revealed that APOB's co-expressed genes were notably clustered in blood microparticle, microbody, dendritic shaft, actin cytoskeleton, and endoplasmic reticulum lumen (Fig. 2E).The analysis of molecular functions (MF) revealed that co-expressed genes showed significant enrichment in molecular adaptor activity, binding to cell adhesion molecules, binding to snRNA, binding to cofactors, and binding to organic acids (Fig. 2F).Based on the examination of KEGG, the co-expressed genes were primarily concentrated in drug processing, pathways related to fatty acids, carbon processing, the cell cycle, and the control of actin cytoskeleton (Fig. 2G).

APOB levels and clinical characteristics for HCC patients
Through the UALCAN online tool, we evaluated APOB levels among various patient groups based on several parameters.Compared with normal controls, APOB levels were markedly decreased in both females and males with HCC (Fig. 3A).With regards to cancer stage, APOB levels were lower in HCC patients classified as stages 1,2, 3 and 4 (Fig. 3B).Based on tumor stage, a marked decrease in APOB levels was detected in HCC patients in grades 2, 3 and 4 (Fig. 3C).As for nodal metastasis, APOB levels were lower in HCC patients classified as N0 (Fig. 3D).Regarding age, there was a significant decline in APOB levels in HCC patients in aged 41-60 years and 61-80 years (Fig. 3E).APOB expression was dramatically decreased in HCC patients of african-american, caucasian and asian origin (Fig. 3F).In addition, Down-regulation of APOB levels were observed in both TP53 wild-type and TP53-mutant in HCC patients (Fig. 3G).

Analysis and prediction of miRNAs associated with APOB
NcRNAs are vital in gene expression regulation.To investigate the potential influence of various ncRNAs on APOB, we employed the starbase database to predict miRNAs that could potentially interact with APOB.As a result, six miRNAs were identified (Table 1).In the context of HCC, APOB exhibited a significant inverse correlation with hsa-miR-21-5p, hsa-miR-9-5p, and hsa-miR-877-5p while displaying a favorable correlation with hsa-miR-505-3p.Subsequently, we conducted a comprehensive analysis to assess the expression levels and prognostic relevance of these four miRNAs in HCC.The expression of Hsa-miR-21-5p, hsa-miR-9-5p, and hsa-miR-877-5p was significantly increased in HCCs, and reducing their levels was linked to favorable patient outcomes (Fig. 5).

Correlation between APOB and immune checkpoints expressions in HCC
The immune escape of tumors is significantly influenced by immune checkpoints such as CTLA4, PDCD1 and CD274.To assess the potential tumor suppressor role of APOB in HCC, we employed the Spearman correlation coefficient to evaluate the relationship between APOB and CD274, PDCD1, and CTLA4.After adjusting for purity, a significant positive correlation was observed between APOB expression and CD274 in patients with HCC, as depicted in Fig. 9B-D.Conversely, a negative association was observed between APOB expression and PDCD1 as well as CTLA4.In HCC patients, we found significant inverse associations between APOB and PDCD1 as well as CTLA4, similar to the findings from GEPIA data analysis (Supplementary Fig. 3B-C).

Dicussion
At present, HCC is strongly linked to unfavorable prognostic results.Discovering the molecular mechanisms that cause HCC carcinogenesis could potentially enhance the development of therapeutic targets and identify valuable prognostic biomarkers.APOB plays a major role in HCC.Nevertheless, APOB's significance in HCC should be further explored.The expression of APOB in HCC from the TCGA project was assessed.APOB levels were verified using the UALCAN and TIMER 2.0 databases.Afterwards, we investigated the co-expressed genes of APOB and conducted enrichment analyses using LinkedOmics.The survival analysis for APOB demonstrated that HCC individuals exhibiting reduced APOB levels experienced unfavorable prognostic results.The authors Lee and colleagues [32] conducted a study of 30 individuals and revealed that patients diagnosed with HCC and with deactivated APOB experienced worse results.These results show the inhibitive role of APOB in HCC.
Currently, the exact mechanism behind the correlation between APOB and HCC is still unidentified.However, individuals diagnosed with familial hypobetalipoproteinemia (FHBL) have previously demonstrated the presence of APOB deleterious mutations, which are linked to reducedlevels of low-density lipoprotein cholesteroland overall cholesterol [57].Individuals with FHBL caused by APOB mutations will experience the development of liver cirrhosis, hepatocarcinoma, and hepatic steatosis [58].
Radiotherapy, chemotherapy, and immunotherapy can be affected by tumor immune cell infiltrations, impacting prognostic outcomes for cancer patients [73][74][75].Our findings demonstrated that APOB is markedly negatively associated with B cells, dendritic cells and CD4 + T cells in HCC.These findings suggest that tumor immune infiltrations could contribute to the inhibitory effect of APOB on HCC.

Fig. 1
Fig. 1 Expression of APOB in HCC.A TIMER was used to examine APOB expression in a variety of cancer types (B) mRNA expression of APOB in HCC tissues and adjacent normal liver tissues (UALCAN).C immunohistochemical images of APOB in HCC tissues and normal liver tissues (Human Protein Atlas).Note: * p < 0.05, ** p < 0.01,*** p < 0.001

Fig. 2 Fig. 3
Fig. 2 Enrichment analyses of APOB co-expressed genes.APOB co-expressed genes was display by volcano map (A), Heat map of top 50 positively (B) and 50 negatively (C).GO enrichment analysis for biological processes (D), cellular components (E), molecular functions (F) and KEGG (G)

Fig. 4
Fig. 4 APOB's prognostic value was assessed by the Kaplan-Meier plot.A-D The Kaplan-Meier plot is shown for OS (A), PFS (B), RFS (C) and DSS (D).A forest plot shows the correlation between APOB expression and clinicopathological parameters in HCC patients (Kaplan-Meier Plotter) (E-F)

Table 1
The expression correlation between predicted miRNAs and APOB in HCC analyzed by starBase database